Analysis and Characterization of Cultivable Extremophilic Hydrolytic

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE Analysis and characterization of cultivable extremophilic hydrolytic bacterial community in heavy-metal- contaminated soils from the Atacama Desert and their biotechnological potentials M.L. Moreno1, F. Piubeli2, M.R.L. Bonfa´ 2, M.T. Garcı´a1, L.R. Durrant2 and E. Mellado1

1 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Sevilla, Spain 2 Departamento de Ciencia^ de Alimentos–FEA Universidade Estadual de Campinas-UNICAMP, Campinas, SP Brazil

Keywords Abstract Atacama Desert, halophiles, halotolerants, heavy-metal-contaminated soils, hydrolytic Aims: To isolate and characterize the cultivable community of hydrolase extremophilic bacteria. producers (amylase, protease, lipase, DNase, xylanase and pullulanase) inhabiting heavy-metal-contaminated soils in extreme conditions from the Atacama Desert. Correspondence Methods and Results: A total of 25 bacterial strains showing hydrolytic Encarnacio´ n Mellado, Department of Microbi- activities have been selected including halotolerants, extremely halotolerants ology and Parasitology, Faculty of Pharmacy, and moderate halophiles. Most hydrolase producers were assigned to the University of Sevilla, C/Profesor Garcıá Gonza´ lez, 2, 41012 Sevilla, Spain. E-mail: family Bacillaceae, belonging to the genera Bacillus (nine strains), Halobacillus [email protected] (seven strains) and Thalassobacillus (five strains) and four isolates were related to members of the families Pseudomonadaceae, Halomonadaceae and 2012/0577: received 30 March 2012, revised Staphylococcaceae. The selected strains were then characterized for their 12 June 2012 and accepted 16 June 2012 tolerance pattern to six heavy metals, measured as minimal inhibitory concentrations (MICs). doi:10.1111/j.1365-2672.2012.05366.x Conclusions: The diversity found in the cultivable bacterial community analysed is more limited than that detected in other ecological studies owing to the restrictive conditions used in the screening. The dominant bacteria were Firmicutes and particularly, species related to the genus Bacillus. Significance and Impact of the Study: This study is focused on the characterization of extremophilic hydrolytic bacteria, providing candidates as a source of novel enzymes with biotechnological applications.

organic species detected in the soils (Navarro-Gonza´lez Introduction et al. 2003). In spite of the extreme conditions of this The Atacama Desert in northern Chile is a coastal desert desert habitat, a broad spectrum of halotolerant and usually described as one of the driest deserts on Earth, halophilic bacteria and archaeas have been found in salt- with a surface that has been minimally disturbed by natu- flats and hypersaline lakes in the area (Demergasso et al. ral erosion for millions of years (McKay et al. 2003; 2004; Wierzchos et al. 2006; Connon et al. 2007; De los Hartley et al. 2005). This region includes arid and semi Rıós et al. 2010). arid environments with many different saline deposits. In Different classification schemes have been designed to these saline deposits, evaporative basins called saltflats define the relationships of micro-organisms with salt. The can be found (Chong 1984) containing a high percentage most widely accepted classification is based on the of salts from the leaching of volcanic rocks. optimal salt concentration for their growth (Kushner and Until recently, regions of the Atacama Desert were Kamekura 1988). considered the dry limit of photosynthetic activity and The halotolerance of many enzymes derived from primary production (Warren-Rhodes et al. 2006), with halotolerant and halophilic micro-organisms can be extremely low levels of cultivable organisms and oxidized exploited wherever enzymatic transformations are

© 2012 The Authors 550 Journal of Applied Microbiology 113, 550--559 © 2012 The Society for Applied Microbiology M.L. Moreno et al. Extremophilic hydrolytic bacterial community required to function under extreme physical and chemical annual precipitation in AS and DV is <1 and 4 mm, conditions (Oren 2002a; Setati 2010). Hydrolases respectively, with a high UV index. At each site, we col- produced by halophiles have quite diverse potential usage lected four samples, including pieces of halite crusts bro- in different areas (food industry, feed additive, biomedi- ken off AS and surface soils (0–20 mm) from DV. cal sciences and chemical industries) (Rao et al. 1998; Samples were taken in sterile plastic containers and stored Kulkarni et al. 1999; Niehaus et al. 1999) and several in an icebox until further processed. Concentrations of halophilic enzymes have been tested for their potential Na, Ca, K, Fe, Mg, B, As, Mn, Cu and Zn were deter- biotechnological applications, including amylases, mined via ICP-OES (Inductively coupled plasma-optical nucleases and proteases (Oren 2002b). emission spectrometry) using an Iris Advantage spectrom- The Atacama Desert has increased its levels of eter (Thermo Jarrel Ash Corporation, MA, Franklin, heavy metals owing to natural and industrial processes USA). Calcium carbonate (CaCO3) was determined using (Salamanca et al. 2000). Cadmium, copper and zinc are an automated continuous flow analyser (Bran + Luebbe among those heavy metals that are being released in the III, Germany). Soil pH and electrical conductivity (EC) environment. Although some heavy metals constitute were determined in a 1 : 5 soil/water extract. An essential trace elements, most can be, at high concentra- electronic thermometer (Orion model 290) was used to tions, toxic to organisms from all branches of life, includ- measure temperature. Soil samples were air-dried, crushed ing bacteria (Nies 1999). However, some bacteria have and sieved through a 2-mm sieve. Total organic carbon adapted their physiology to tolerate the presence of metals (TOC) was analysed by dichromate oxidation and titra- or can even use them to grow. Owing to their exceptional tion with ferrous ammonium sulphate (Walkley and Black characteristics, salt tolerant bacteria adapted to live in 1934). hostile environments may exhibit the potential to remove heavy metals from contaminated environments (Mishra Isolation of hydrolytic producers et al. 2009; Zhuang et al. 2010). However, very little information is available on the heavy-metal tolerance of The isolation of micro-organisms producing hydrolytic bacteria in hypersaline environments, with the exception enzymes was performed by diluting collected samples À À of a few studies of metal-resistant halophilic bacteria from the Atacama Desert (from 10 1 to 10 3) in saline isolated from different habitats such as the Maruit Lake, solution (0Á9% NaCl, w/v) and inoculating 100 ll of the Egypt (Osman et al. 2010) or the Dead Sea Shore, Jordan dilution into saline medium (BS-10 medium) with 10% (Amoozegar et al. 2005; Massadeh et al. 2005). (w/v) total salts and 1% (w/v) yeast extract and substrates Therefore, in the present work, we studied the cultiva- to assay for amylase, protease, lipase, DNase, xylanase ble diversity of hydrolytic enzyme producers in a bacterial and pullulanase activities. The salts solution (SW) community from saline soils of the Atacama Desert. Fur- composition contained in the BS medium was as follows À1 thermore, these strains were evaluated for their biotech- (g l ): NaCl (234), MgSO4Á7H2O (61), MgCl2Á6H2O nological potentials in terms of hydrolytic enzyme (39), KCl (6), CaCl2 (1), NaBr (0Á7) and NaCO3H(0Á2). activity and heavy-metal tolerance. When necessary, different concentrations of salts solution were added to BS medium as indicated. Solid media were prepared by adding 2% (w/v) bacteriological-agar (Difco Materials and methods Laboratories, Detroit, MI). The plates were incubated at 37°C in aerobic conditions. Description of sampling sites and collection Amylase activity was determined using solid BS-10 med- Samples were collected in September 2010 from two ium supplemented with 0Á5% (w/v) soluble starch. After different points in the Atacama Desert region (Chile), des- incubation for 15 days, the plates were flooded with 0Á3% ignated as follows: Atacama Salar (AS) (in the Pre-Andean (w/v) I2 À0Á6% (v/v) KI solution: a clear zone around the Depression) (latitude 23º300′S, longitude 68º150′W) and colonies indicated hydrolysis of starch (Cowan and Steel Death Valley (DV), (latitude 22º555′S, longitude 68º120′ 1982). Protease activity was screened qualitatively in the W), both closely located to copper and lead–zinc mine saline BS-10 medium amended with 2% (w/v) skim milk. tailings. The AS is the largest saltflat in Chile and is Zones of precipitation of paracasein around the colonies located in the east region of the Antofagasta district of appearing over the next 15 days were taken as evidence of Chile. The Salar is 100 km long and 80 km wide, and the proteolytic activity (Cowan and Steel 1982). Lipase activity salts have an athalassohaline origin. The DV, also known of the strains was detected by screening for zones of hydro- as the Mars Valley, located in the Salt Mountain range, is lysis around colonies growing on solid BS-10 medium, very close to San Pedro de Atacama, standing out owing containing 1% (w/v) tributyrin after incubation for to its sand dunes of up to 150 m in height. The average 15 days (Mourey and Kilbertus 1976). The presence of

DNase activity on plates was determined on DNase test gene was amplified by polymerase chain reaction (PCR) agar (BBL) containing 10% (w/v) total salts. After incuba- using total DNA as the template and universal primers tion for 15 days, the plates were flooded with 1 N HCl designed for Bacteria. The 16S rRNA PCR amplification solution. Clear halos around the colonies indicated DNase was performed using the forward primer 16F27 (5′-AGA- activity (Jeffries et al. 1957). Xylanase and pullulanase GTTTGATCMTGGCTCAG-3′) and the reverse primer activities were tested in BS-10 medium with the chromo- 16R1488 (5′-CGGTTACCTTGTTAGGACTTCACC-3′)(Lane genic substrates (0Á1%, w/v) AZCL-xylan and AZCL-pullu- 1991). The following programme was used for amplifica- lan (Megazyme, Bray, Ireland). After this time, xylanolytic tion: one cycle of 95°C for 5 min, 25 cycles of 94°C for and pullulolytic activities were detected showing clearing 1 min, 50°C for 1 min, and 72°C for 2 min, and then an zones around the colonies. extension step for 10 min at 72°C. The amplified DNA The positive hydrolytic isolates (64 strains) were fragments were analysed on 1% (w/v) agarose gels stained replicated in the solid BS-10 medium containing the cor- with ethidium bromide and photographed with UV responding substrates and were tested again for the illumination. hydrolytic activities. A Gram stain test of the colonies was performed before proceeding to further studies. Amplified rDNA restriction analysis (ARDRA) and 16S rRNA sequence analysis Salt requirement The PCR products of the 16S rRNA gene were digested The growth response to NaCl of the isolates was with 1 U of the restriction endonucleases AluI and HhaI examined in BS medium containing 0, 1, 3, 5, 7Á5, 10, (Fermentas MBI, Vilnius, Lithuania) at 37°C for 2 h. The 12Á5, 15, 17Á5, 20, 25 and 30% (w/v) NaCl and incubated digestion products were detected by gel electrophoresis in at 37°C on a rotary shaker (New Brunswick Scientific Co, 2% agarose gels and visualized after staining with ethidium À NJ, USA) operating at 200 rev min 1. Growth curves of bromide. ARDRA profiles were analysed, and representa- the isolates were obtained by monitoring the culture tive unique clones were selected for nucleotide sequence absorbance at 600 nm using a Perkin-Elmer spectropho- determination. The amplified DNA fragments were tometer at different incubation times. For this purpose, sequenced using an automated DNA sequencer model 250-ml flasks containing 50 ml of saline medium were 9700 (Applied Biosystems, Foster City, CA). The 16S each inoculated with 100 ll of a stationary-phase culture. rRNA gene sequences obtained were compared to the Samples (500 ll) were taken at 6, 12, 18, 24, 40 and 48 h sequences in the GenBank data bases and aligned with to monitor the culture absorbance. the most similar sequences available to construct the phylogenetic trees using the ARB software package (Ludwig and Strunk 1996). Evolutionary distance matrices were Heavy-metal tolerance testing calculated using the neighbour-joining algorithm (Ludwig For the determination of tolerance to heavy metals, solid et al. 1998). Phylogenetic trees using different methods BS medium containing the optimal concentration of (distance matrix, maximum parsimony and maximum NaCl for each isolate was used. The range of concentra- likelihood) were constructed and compared to elucidate tions for the heavy metals tested (cadmium, zinc, nickel, the confidence of local topologies. À iron, copper and cobalt) ranged from 0Á5 to 4 mmol l 1 À (0Á5, 1, 2, 3 and 4 mmol l 1). Controls consisting of Nucleotide sequence accession numbers media without metals and inoculated with the test micro- organisms were carried out in all experiments. After The nucleotide sequences reported in this work have been incubation at 37°C for 2–3 days, the results were inter- deposited in the EMBL Nucleotide Sequence Database preted. The lowest concentration of metal that completely under accession numbers HE586566 to HE586590. prevented growth was named the minimum inhibitory concentration (MIC). The MICs for all strains were Results determined by triplicate. Physicochemical analysis of the collected samples Isolation of genomic DNA and amplification of 16S Both sampling areas selected in the Atacama Desert are rRNA gene characterized by a high solar radiation and large tempera- DNA of isolated strains was extracted and precipitated ture fluctuations between day and night. following the CTAB protocol for bacterial genomic DNA Table 1 shows the physicochemical characterization of preparations (Wilson 1987). The 16S ribosomal RNA the collected samples. The main ionic composition in

Table 1 Chemical and physical properties of collected samples from isolates from DV). Concerning the pullulanase producers the Atacama Desert region (17 strains), this group represents the 17% of the Isolation site hydrolytic population (15 isolates from AS and two isolates from DV). In the analysed community, the prote- Parameter Atacama Salar (AS) Death Valley (DV) olytic producers constituted 15% of the isolates showing Na+ (mg lÀ1)* 1648 222 hydrolytic activities (10 isolates from AS and ﬁve isolates Ca2+ (mg lÀ1)* 184 233 from DV). Xylanases were the least hydrolases repre- K+ (mg lÀ1)* 163 214 sented (eight isolates from AS and three isolates from À Fe (mg l 1)* 29Á6 195 DV) (Table 2). Combined activities were detected in 2+ À1 Á Mg (mg l )* 196 59 43 most of the isolates. Eleven strains presented ﬁve hydro- B (mg lÀ1)* 51Á91 0Á311 À lytic activities, nine strains presented four hydrolytic As (mg l 1)* 1Á194 0Á276 Mn (mg lÀ1)* 35Á84 0Á05 activities, two strains presented three hydrolytic activities Cu (mg lÀ1)* 3Á13 0Á017 and three strains presented two hydrolytic activities. No Zn (mg lÀ1)* 3Á972 <0Á001 strains with all of the hydrolytic activities tested have pH 8Á68 ± 0Á064 9Á54 ± 0Á11 been found in this study. EC (mS cmÀ1) 127 ± 11Á30Á633 ± 0Á0133 TOC (mg kgÀ1) <0Á10Á91 ± 0Á01 Moisture (%) 7Á30 3Á50 Determination of the saline requirement

Mean values ± standard deviation (n = 3) of Moisture content, Electri- The assay performed to determine the salt growth profile cal Conductivity (EC), pH and Total Organic Carbon (TOC) in samples. for the 25 isolates (Table S1, supporting information) *Average values of the collected samples from two sampling sites. revealed that most of them grew in the NaCl range of 2–25% (w/v) and showed optimal growth at 7Á5% (w/v) both sampling sites was different. Samples from AS NaCl. Seven strains showed optimal growth without presented higher salt concentrations, particularly Na+ NaCl, although they were able to grow in presence followed by Mg+,Ca+,K+ and Mn+ ions. The analysis of NaCl and other salts. Only two strains showed optimal of samples from DV revealed a similar concentration growth without NaCl but were able to grow at high of Ca+ and K+ ions but significantly higher concentra- concentrations of salts (up to 17Á5% NaCl). tions of Fe. The pH values differed in AS and DV although in both cases alkaline values were obtained. The À Phylogenetic analysis of the isolates TOC content was lower in AS (<0Á1mgkg 1) than DV À (0Á91 mg kg 1). Sodium concentration and electrical Comparative sequence analyses of the 16S rRNA genes conductivity displayed the largest difference along our from the 25 isolates were performed (Table 2). The study samples. majority of the isolates were Gram-positive bacteria assigned to the family Bacillaceae and included in the genera Bacillus (nine strains), Halobacillus (seven strains) Screening and selection of extremophilic and Thalassobacillus (five strains). Two Gram-negative micro-organisms producing hydrolytic enzymes isolates were assigned as members of the Pseudomonada- A total of 64 colonies showing hydrolytic activity were ceae and Halomonadaceae families, included in the genera chosen for analysis. The 16S rRNA genes of these isolated Pseudomonas and Halomonas, respectively. Only two strains were amplified. Restriction analysis of the ampli- species were detected in the study belonging to the genus fied 16S rRNA genes using the restriction endonucleases Salinicoccus within the Staphylococcaceae family. Essen- AluI and HhaI indicated the presence of different groups tially, similar results were obtained using different meth- of restriction patterns. Twenty-five unique isolates repre- ods to construct the phylogenetic trees. Therefore, we senting the major cluster groups were selected for further only include the neighbour-joining tree (Fig. 1). analysis. Among the hydrolytic community analysed, the strains The most frequent hydrolytic activity detected in our isolated from the AS samples, containing higher salt study was DNase (20% of the hydrolytic population), concentrations were assigned to five different genera. followed by amylase and lipase activities. From the 25 However, the strains isolated from DV samples, with selected isolates by full 16S rRNA gene sequencing, 21 of moderate salinity and organic matter content, but pre- them showed DNase activity (16 isolates from AS and senting alkaline pH, were all identified as species of the five isolates from DV), 20 showed amylase activity (16 genus Bacillus. isolates from AS and four isolates from DV) and 19 Table 2 summarizes the hydrolytic activities of the iso- showed lipase activity (14 isolates from AS and five lates. The DNase producers represented the group with

Table 2 Bacterial diversity based on the 16S rRNA gene and hydrolytic activities of the strains isolated from the desert soils samples

Hydrolytic activity

Strain Closest relative % Similarity Accession no. Protease Lipase Amylase DNase Pullulanase Xylanase

DV1 Bacillus seohaeanensis 99 AY667495 + ÀÀ + ÀÀ DV2 ++À + ÀÀ DV3 À + À + ÀÀ DV4 Bacillus subtilis subsp. inaquosorum 99 EU138467 +++À ++ AS1 ++À + À + DV5 Bacillus stratosphericus 99 AJ831841 ÀÀ+++ À DV6 Bacillus oceanisediminis 99 GQ292772 ++++À + AS2 Bacillus mojavensis 99 EU138460 À + À + ÀÀ DV7 Bacillus licheniformis 97 AB301007 +++ÀÀ + AS3 Pseudomonas halophila 99 AB021383 ++++ÀÀ AS4 Halobacillus hunanensis 98 FJ425898 +++++ À AS5 +++++ À AS6 Halobacillus profundi 98–99 AB189298 ÀÀ+++ + AS7 ÀÀ+++ + AS8 À ++ ++ + AS9 ÀÀ+++ + AS10 ÀÀ+++ + AS11 Thalassobacillus devorans 98–99 AJ717299 À ++ ++ À AS12 À ++ ++ À AS13 +++++ À AS14 +++++ À AS15 +++++ À AS16 Halomonas organivorans 98 NR_029029 +++++ À AS17 Salinicoccus roseus 98 NR_026311 +++À ++ AS18 +++À ++ the highest number of strains (21 strains), including Response to heavy metals isolates with high affinity to the genera Bacillus, Haloba- cillus, Thalassobacillus and closely related to the species The heavy-metal tolerance levels of the isolates from the Pseudomonas halophila and Halomonas organivorans. Atacama Desert to six heavy metals, expressed as MICs, Among the amylolytic strains (20 strains), most of them are shown in Table 3. There is not a specific and were related to moderate halophiles included in the accepted metal concentration that could be used as a genera Halobacillus, Thalassobacillus, Salinicoccus, Halo- standard to define universally metal resistance. Moreover, monas and Pseudomonas. Only four strains were closely metal salts and microbial media components can interact related to halotolerant members of the genus Bacillus. in ways which make data interpretation difficult. Thus, in Isolates producing lipolytic enzymes (19 strains) showed general, for the metal tested, those strains which were not À high affinity to species related to the genera Bacillus, inhibited by 4Á0 mmol l 1 of the metal ions could be Pseudomonas, Halobacillus, Thalassobacillus, Halomonas considered tolerant to these metals. The strains that were À and Salinicoccus. The presence of lipolytic activity was an not inhibited by 3Á0 mmol l 1 could be considered important characteristic to differentiate isolates assigned moderately tolerant to these metals. to the same species (Bacillus seohaeanensis, Halobacillus On the basis of the MICs, the highest toxicities were hunanensis and Halobacillus profundi). A total of 17 found with cadmium and zinc. Only one strain related to strains showed pullulanase activity. In comparison with Thalasobacillus devorans (AS14) was cadmium tolerant. other activities, an absence of pullulanase activity was Three strains, related to the genera Bacillus (DV4 and found among the Bacillus strains. Concerning the isolates DV5) and Thalassobacillus (AS12) could be considered as producing proteolytic enzymes (15 strains), we detected moderately tolerant to cadmium. Only two strains related strains related to the genera Bacillus, Pseudomonas and to the genus Bacillus (DV1 and DV6) were considered Halomonas and closely related to Thalassobacillus devo- tolerant to zinc and three strains (DV3, DV4 and DV5) rans, H. hunanensis and Salinicoccus roseus. Only 11 as moderately zinc tolerant. In contrast, Ni was shown to strains showed xylanase activity and were included in the be the least toxic metal for the isolates, with 56% of the genera Halobacillus, Bacillus and Salinicoccus. tested isolates demonstrating tolerance to this metal. The

74 AS1 74 AS2 57 Bacillus mojavensis NRRL 14698T 74 DV4 81 Bacillus subtilis subsp. inaquosorum KCTC 13429T 100 Bacillus licheniformis ATCC 14580T 68 DV7 100 DV5 Bacillus stratosphericus JCM 13349T DV2 DV1 Bacillus seohaeanensis DSM 16464T 65 DV3 71 DV6 85 Bacillus oceanisediminis JCM 16506T 100 Bacillus firmus ATCC 14575T 98 Bacillus infantis JCM 13438T 65 AS6 AS7 89 AS10 66 AS9 96 Halobacillus profundi DSM 18394T AS8 83 Halobacillus Karajiensis DSM 14948T AS4 100 Halobacillus hunanensis DSM 21184T 100 AS5 AS14 74 AS13 Thalassobacillus devorans CECT 7046T AS11 100 AS12 96 AS15 100 Bacillus alcalophilus JCM 5262T 100 AS17 100 AS18 100 Salinicoccus roseus ATCC 49258T 89 Halomonas organivorans CECT 5995T 100 AS16 100 Halomonas salicampi KCTC 12127T 52 Pseudomonas halophila DSM 3050T 100 Halovibrio denitrificans DSM 15505T AS3 Streptomyces aureofaciens NRRL 2209T

Figure 1 Phylogenetic neighbour-joining tree based on the analysis of 16S rRNA gene sequences, showing the diversity of micro-organisms able to produce hydrolytic enzymes. The isolates described in the present study are shown in bold. Isolates from Atacama Salar are indicated as AS, and isolates from Death Valley are indicated as DV. 16S rRNA gene sequences from the isolates correspond to sequences of 1400 bp. Bootstrap values (>50%, based on 1000 replications) are shown at branch points. Bar represents a 2% sequence difference. susceptibility levels of the isolated strains to Fe and Cu Discussion was more heterogeneous, nine strains (36% of the total isolates) were tolerant to iron and one strain was moder- Few environmental studies have focused on the study of ately tolerant. Seven strains (DV2, DV4, DV5, AS7, AS9, the bacterial community inhabiting saline and hypersaline AS13 and AS15) were tolerant to copper and two strains habitats in the Atacama Desert (Demergasso et al. 2004; (AS11 and AS14) were moderately tolerant to this metal. Connon et al. 2007; Lester et al. 2007; Mishra et al. 2009; Species related to H. hunanensis (AS4 and AS5) were the De los Rı´os et al. 2010) and, in particular, to our most sensitive to all the metal tested. In contrast, the knowledge, no screening programmes have been designed strains related to Bacillus subtilis (DV4) and Bacillus to select extremophilic micro-organisms with the ability stratosphericus (DV5) were the most tolerant to the heavy to produce enzymes that hydrolyse polymeric com- metals tested, showing higher tolerance to four different pounds, although the hydrolases play a key role in the metal ions (Fe, Co, Ni and Cu) and moderate tolerance geochemical cycling of nutrients as the hydrolysis of high to Cd and Zn. molecular weight biopolymers constitutes an initial step

Table 3 Minimal inhibitory concentrations (MICs) of the metal ions community of moderate halophiles isolated from salterns tested against the bacterial hydrolase producers in Spain, and they described amylase producers as the MIC (mmol lÀ1) most abundant isolates. Furthermore, Moreno et al. (2009) studied the diversity of extreme halophiles, pro- Strain Cd Zn Ni Fe Cu Co ducing lipase, protease, amylase and nuclease in hypersa- DV1 0Á54420Á50Á5 line ecosystems in South Spain, concluding that 70% of DV2 0Á514440Á5 total of the hydrolytic isolates were also amylase produc- DV3 0Á534210Á5 ers. However, Baati et al. (2010) in the screening DV4334444performed in salt mines of Sfax (Tunisia) detected a Á Á AS1 0 5054221similar percentage of proteases, amylases and DNases DV5334444producers. Rohban et al. (2009) investigated the ability of DV6 0Á544411 AS2 0Á50Á54211halophilic strains isolated from a hypersaline lake in Iran DV7 0Á50Á54314to produce nine different extracellular hydrolases (inulin- AS3 0Á50Á51 0Á50Á50Á5 ase, pectinase, cellulase and xylanase). In contrast to our AS4 0Á50Á50Á50Á50Á50Á5 results, the most frequent activity expressed by these AS5 0Á50Á50Á50Á50Á50Á5 isolates was lipase, and the least prevalent activity Á Á Á Á AS6 0 5051 05054represented was DNase. Á Á Á AS7 0 5053 054 4 It is interesting to emphasize that combined hydrolytic AS8 0Á50Á5140Á50Á5 AS9 0Á50Á51 0Á54 2activity was frequently detected in isolates from both AS10 0Á50Á5310Á52sampled areas, AS and the Death Valley. These results AS11 0Á50Á54431support previous studies in other hypersaline habitats AS12 3 0Á5140Á52(Sańchez-Porro et al. 2003; Moreno et al. 2009), although AS13 0Á50Á51 0Á54 2the number of strains showing combined hydrolytic Á AS14 4 0 54432activity is higher in the present study. Á Á AS15 0 522054 3 Based on the comparison of partial sequences of 16S AS16 0Á50Á54424 AS17 0Á5120Á52 1rRNA genes, most environmental isolates able to produce AS18 0Á50Á52 0Á52 1hydrolytic enzymes were Gram-positive bacteria, assigned to the family Bacillaceae, comprising species of the genera Bacillus (Cohn 1872), Halobacillus (Spring et al. 1996) in the metabolism of organic compounds in the different and Thalassobacillus (Garcıá et al. 2005). Bacillus is well ecosystems. known as an extracellular enzymes producer and many Under the restrictive conditions used in our screening, industrial processes use species of this genus for commer- a total of 64 fresh cultures showing hydrolytic activities cial production of enzymes (Schallmey et al. 2004). Only were isolated and from them, 25 strains were selected and two isolates were related to the Gram-negative bacteria characterized. Most isolates (16 strains) grow optimally Ps. halophila (Sorokin et al. 2006) and H. organivorans in media containing between 5 and 7% (w/v) total salts, (Garcıá et al. 2004). The other characterized isolates were being able to grow in most cases up to 25% (w/v) salts. related to S. roseus (Ventosa et al. 1990). Thus, they can be classified as moderate halophiles Although no organic matter is detected in the samples according to the classification proposed by Kushner and obtained from the AS, a broader diversity is found Kamekura (1988). All the moderate halophiles were iso- among the isolates if compared to that obtained from the lated from the AS. The number of halotolerant isolates Dead Valley, identifying only hydrolytic producers from was remarkably lower compared with that of moderate the genus Bacillus. Probably, the presence of hydrolases is halophiles. Representative members of the halotolerant more significant among the population found in the AS group were detected in both sampling sites, and the two samples to obtain nutrients in an adverse environment. extremely halotolerant strains (DV2 and AS2) were The cultivable microbial diversity found in this study is isolated from the Death Valley. more limited than that detected in other ecological The most frequent hydrolytic activity detected in our studies performed in diverse areas of the Atacama Desert, study was DNase, followed by amylase and lipase activi- selecting only strains of the phyla c-Proteobacteria ties. In the analysed community, we also detected pullu- (families Halomonadaceae and Pseudomonadaceae) and lanase and protease producers. Xylanases were the least Firmicutes (families Bacillaceae and Staphylococcaceae). represented hydrolases. Sańchez-Porro et al. (2003) However, this study was carried out under more showed the abundance of five hydrolytic enzymes includ- restrictive conditions. Our results differ from others in ing amylase, protease, lipase, DNase and pullulanase in a the preponderant hydrolytic bacteria. While the overall

© 2012 The Authors 556 Journal of Applied Microbiology 113, 550--559 © 2012 The Society for Applied Microbiology M.L. Moreno et al. Extremophilic hydrolytic bacterial community preponderant bacteria in our study were Firmicutes Acknowledgements (92%), c and b-Proteobacteria were reported as predominant in other studies (Rohban et al. 2009; Baati et al. This work was supported by grants from the Spanish 2010) in which Firmicutes did not appear or they were a Ministry of Science and Education (CTM 2006-03310) minority. and Junta de Andalucıá (P08-RMN-3515). Maria de Several studies have demonstrated that metal toxicity Lourdes Moreno was supported by a fellowship from the can be heavily influenced by environmental factors such University of Sevilla. Francine Piubeli was supported by a as pH, temperature, soluble organic matter, clay minerals, fellowship from Coordination of Improvement of Higher inorganic anionic and cationic components (Nieto et al. Education Personnel (Capes). 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